We have identified a useful tumor-induced target concentrated in specialized vesicular transporters at the endothelial cell surface called caveolae. It is a post-translationally modified form of Annexin A1 (AnnA1) that is sufficiently specific and accessible to enable tumor-selective delivery after intravenous injection of a newly created monoclonal antibody that is specific for AnnA1. Caveolae rapidly pump this antibody with attached cargo across the endothelium to reach concentrations actually inside solid tumors that greatly exceed maximum blood levels. This project focuses on testing the therapeutic utility of the first antibody to target tumor endothelial caveolae and to actively penetrate solid tumors. It evaluates proof-of-principle of our innovative delivery strategy for therapy and moves toward clinical translation by assessing how well caveolae immunotargeting and pumping into tumors can enhance the therapeutic impact of chemotherapy and radiotherapy. Various radionuclides and chemotherapeutic agents will be conjugated to AnnA1 antibody to create new tumor caveolae-targeted therapeutics that will be tested for enhanced delivery and therapeutic efficacy in breast cancer models using mouse and human tissues. We hypothesize that by pumping armed antibodies into tumors, caveolae can rapidly and specifically concentrate therapeutic agents inside tumors to enhance tumor destruction and reduce toxic exposure elsewhere in the body. This novel caveolae targeting strategy should reduce dosages required for efficacy and increase therapeutic indices. The Specific Aims of this Project are: Aim 1. To investigate the delivery and efficacy of systemic radioimmunotherapies targeting caveolae in rodent mammary tumor models. Aim 2. To investigate the utility of caveolae immunotargeting to enhance mammary tumor delivery and efficacy of chemotherapeutic agents in rodent models. Aim 3. To assess targeting, tissue processing and therapeutic efficacy of naked and armed antibodies to human mAnnA1 in an IVM human breast tumor model with functional human blood vessels. This project will utilize spontaneous mammary tumors from genetically engineered mice as well as our new intravital microscopy (IVM) tumor model system. IVM permits direct visualization of targeting and endothelial processing as well as stroma and tumor cell responses, all of which can be quantified to help provide new insights into therapeutic mechanisms in tumors. Targeting caveolae opens a specific gateway across the restrictive blood:tumor interface. It has advanced delivery much closer to ideal targeting needed to achieve more effective therapies. We have demonstrated that AnnA1 is expressed in the vasculature and caveolae of human primary and metastatic breast tumors. Thus, these studies are likely to yield new delivery platforms to create novel diagnostic imaging agents and tumor targeting therapies for many breast tumors in humans.